Professional Documents
Culture Documents
.
Take drops of egg suspension and put them in a tube. Let
theeggs settletothebottom, or givethemafewgentlespins
with the hand centrifuge. Remove all of the overlying sea-
water and ll the tube with Ca
ions, so the
dilution you are getting on each wash is important. Say you
get a 1:50 dilution on each wash, and you do two washes
into CaFSW. That means that you diluted the original sea-
water by 2,500 times, so the Ca
needed
for the CG reaction does not need to come from the
surrounding seawater.
Testing if the FE has Hardened
The FE will harden in 13 min after it elevates from the
egg surface (depending on the species). Fertilize some
eggs on a slide, and after 5 min add a drop of the powerful
ionic detergent 5% SDS in water (sodium dodecyl sulfate).
The cell should totally dissolve, but leave the FE intact as a
circle. This demonstrates just how hard (chemically stable)
the FE really is. To study the amino acid composition of the
FE, the FEs are isolated in a buffer containing SDS, and the
solubilized proteins washed away with fresh SDS. Then the
FEs are washed into distilled water and nally hydrolyzed
with6NHCl at 100
and H
needed for
exocytosis of the CG is released from the endoplasmic
reticulum of the eggs.
Articial Activation of Sea Urchin Eggs in Procaine
Sea urchin eggs are metabolically repressed because
their internal pHis 6.8. Fiveminutes after insemination, their
internal pHincreases to 7.2 (Epel, 1990). The change in pH
is what activates microtubule polymerization to form
asters, and also activates protein and DNA synthesis.
Local anesthetics, such as procaine-HCl, increase the
internal pH of cells very gently. If sea urchin eggs are put
in 5mM procaine in HSW pH 8.0, they turn on protein and
DNA synthesis and undergo cycles of chromosome con-
densation and decondensation. They also cycle the poly-
merization and depolymerization of microtubules to form
quasi-mitotic spindles (Vacquier and Brandriff, 1975). This
experiment takes hours to overnight. Put some eggs in
HSWcontaining5 mMprocaine. Haveacontrol dishof eggs
in seawater without procaine. Observe the eggs for several
hours. What do you seeafter 610 hr inprocaine/seawater?
Are there changes in the nucleus? Do you see cycles of
microtubule formation in that the structure of the cytoplasm
appears different from the unfertilized condition?
Twinning of Sand Dollar Embryos in Dithiothreitol
This works very well with sand dollar eggs (E. parma or
D. excentricus) because their cleavage stage cells rely
more on cell-to-cell surface interaction to form blastulae
than they do to being contained within a thick hyaline layer.
Sea urchin eggs rely more on the clear hyaline layer on the
egg surface for blastomere interaction to form normal blas-
tulae, and nally gastrulae and plutei. Fertilize some
E. parma or D. excentricus eggs and culture them at
room temperature in a medium size petri dish. They should
begin to divide in 4050 min. Watch themclosely and when
they begin to elongate in early telophase, pour them into a
graduated tube and add an equal volume of 100mMDTT in
HSW. They should nish cytokinesis in 5 min. Ten min after
adding the DTT, settle the embryos by gentle hand cen-
trifugation, remove the DTT/HSWand ll the tube with fresh
HSW. Wash them once by settling or by gentle hand
centrifugation, and then resuspend the two-cell stage
embryos in pure seawater and culture them. Those zygotes
that divided in DTT had the SS bonds on all their surface
proteinsreducedtoSHbonds. Thereducedsurfaces fail to
adhere to each other at the two-cell stage and each of the
cells goes on to form a twin embryo. It is possible to get
100% twins (Vacquier and Mazia, 1968). Separation of
embryocells at thetwo- or four-cell stageis theway identical
twins (monozygotic twins) form in mammals.
Culturing Normal Embryos
Set up culture dishes of normally fertilized A. punctulata,
E. parma, L. pictus, or L. variegates embryos in medium
size petri dishes (5cmdiameter, 7 ml total volume HSW) for
long-termobservations of embryogenesis. Do not put more
than a few hundred eggs in each dish. If you can see the
eggs as less than a monolayer of cells covering the dish
bottom, that is plenty. You can keep these cultures at
1522
C.
Solutions Made in HSW (Chemicals From Sigma,
St. Louis, MO)
India ink or Sumi ink: 10% v/v.
SBTI: 20 ml at 1.5 mg/ml, and 5 ml at 10 mg/ml.
PABA: 20 ml at 100mg/ml.
Procaine-HCl at 5 mM.
DTT: 100ml of 10 mM pH 9.1, 20 ml of 100mM, pH 8.0.
Pancreatic trypsin: 1 mg dissolved in 400ml, and 20 ml at
0.1 mg/ml.
Propionic acid: 50 ml of 100mM.
Solutions Made in Deionized Water
0.5 MKCl.
0.1 and 1 NHCl, 100ml each.
0.1 and 1 NNaOH, 100ml each.
1.0 M Tris pH 8.0, 25 ml.
5% w/v SDS at room temperature.
REFERENCES
Darszon A, Beltran C, Felix R, Nishigaki T, Trevino CL. 2001. Ion
transport in sperm signaling. Dev Biol 240:114.
Darszon A, Acevedo JJ, Galindo BE, Hernandez-Gonzalez EO,
Nishigaki T, Trevino CL, Wood C, Beltran C. 2006. Sperm
channel diversity and functional multiplicity. Reproduction
131:977988.
Darszon A, Guerrero A, Galindo BE, Nishigaki T, Wood CD.
2008. Sperm activating peptides in the regulation of ion
fluxes, signal transduction and motility. Int J Dev Biol 52:595
606.
Endo Y. 1961. Changes in the cortical layer of the sea urchin egg
at fertilization as studied with the electron microscope. I.
Clypeaster japanicus. Exp Cell Res 25:383397.
Epel D. 1990. The initiation of development at fertilization. Cell Diff
Dev 29:113.
Ettensohn CA, Wessel GM, Wray GA. 2004. Development of sea
urchins, ascidians and other invertebrate deuterostomes:
Experimental approaches. Meth Cell Biol 74:1875.
Hirohashi N, Vacquier VD. 2002. Egg sialoglycans increase intra-
cellular pH and potentiate the acrosome reaction of sea urchin
sperm. J Biol Chem 277:80418047.
Jaffe LA. 1976. Fast block to polyspermy in sea urchin eggs is
electrically mediated. Nature 261:6871.
Kaupp UB, Kashikar ND, Weyand I. 2008. Mechanisms of sperm
chemotaxis. Ann Rev Physiol 70:93117.
Kamei N, Glabe CG. 2003. The species-specic egg receptor for
sea urchin sperm adhesion is EBR1, a novel ADAMTS protein.
Genes Dev 17:25022507.
Loeb J. 1899. On the nature of the process of fertilization and the
articial production of normal larvae (plutei) fromthe unfertilized
eggs of the sea urchin. Am J Physiol 3:135138.
Moy GM, Springer SA, Adams SL, Swanson WJ, Vacquier VD.
2008. Extraordinary intraspecic diversity in oyster sperm
bindin. Proc Natl Acad Sci USA 105:19931998.
Schroeder TE. 1986. Echinoderm gametes and embryos. Meth
Cell Biol 27:1470.
Su Y-H, Chen S-H, Zhou H, Vacquier VD. 2005. Tandem mass
spectrometry identies proteins phosphorylated by cyclic AMP-
dependent protein kinase when sea urchin sperm undergo the
acrosome reaction. Dev Biol 285:116125.
Townley IK, Schuyler E, Parker-Gur M, Foltz KR. 2009. Expression
of multiple Src family kinases in sea urchin eggs and their
function in calcium release at fertilization. Dev Biol 327:
465477.
Mol Reprod Dev 78:553564 (2011) 563
LABORATORY ON SEA URCHIN FERTILIZATION
Vacquier VD. 1986. Activation of sea urchin spermatozoa
during fertilization. Trends in Biochemical Sciences 11:77
81.
Vacquier VD. 1998. Evolution of gamete recognition proteins.
Science 281:19951998.
Vacquier VD, Brandriff B. 1975. DNA synthesis in unfertilized
sea urchin eggs can be turned on and turned off by the
addition and removal of procaine hydrochloride. Dev Biol
47:1232.
Vacquier VD, Hirohashi N. 2004. Sea urchin spermatozoa. Meth
Cell Biol 74:524544.
Vacquier VD, Mazia D. 1968. Twinning of sand dollar embryos
by means of dithiothreitol. The structural basis of blastomere
interactions. Exp Cell Res 52:209221.
Vilela-SilvaA-CES, Hirohashi N, MouraoPAS. 2008. Thestructure
of sulfated polysaccharides ensures a carbohydrate-based
mechanism for species recognition during sea urchin fertiliza-
tion. Int J Dev Biol 52:551559.
Wong JL, Wessel GM. 2008. Renovation of the egg extracellular
matrix at fertilization. Int J Dev Biol 52:545550.
Zigler KS. 2008. Theevolution of seaurchinspermbindin. Int J Dev
Biol 52:791796.
564 Mol Reprod Dev 78:553564 (2011)
Molecular Reproduction & Development VACQUIER